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Journal of Bacteriology, September 2005, p. 5868-5876, Vol. 187, No. 17
0021-9193/05/$08.00+0     doi:10.1128/JB.187.17.5868-5876.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Characterization of the Exosporium Basal Layer Protein BxpB of Bacillus anthracis

Christopher T. Steichen, John F. Kearney, and Charles L. Turnbough Jr*

Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294

Received 30 April 2005/ Accepted 3 June 2005


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ABSTRACT
 
Bacillus anthracis spores, the cause of anthrax, are enclosed by a prominent loose-fitting structure called the exosporium. The exosporium is composed of a basal layer and an external hair-like nap. The filaments of the hair-like nap are apparently formed by a single collagen-like glycoprotein called BclA, whereas several different proteins form or are tightly associated with the basal layer. In this study, we used immunogold electron microscopy to demonstrate that BxpB (also called ExsF) is a component of the exosporium basal layer. Binding to the basal layer by an anti-BxpB monoclonal antibody was greatly increased by the loss of BclA. We found that BxpB and BclA are part of a stable complex that appears to include the putative basal layer protein ExsY and possibly other proteins. Previous results suggested that BxpB was glycosylated; however, our results indicate that it is not a glycoprotein. We showed that {Delta}bxpB spores, which lack BxpB, contain an exosporium devoid of hair-like nap even though the {Delta}bxpB strain produces normal levels of BclA. These results indicated that BxpB is required for the attachment of BclA to the exosporium. Finally, we found that the efficiency of production of {Delta}bxpB spores and their resistance properties were similar to those of wild-type spores. However, {Delta}bxpB spores germinate faster than wild-type spores, indicating that BxpB suppresses germination. This effect did not appear to be related to the absence from {Delta}bxpB spores of a hair-like nap or of enzymes that degrade germinants.


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INTRODUCTION
 
Bacillus anthracis is a gram-positive, rod-shaped, aerobic bacterium that causes anthrax in humans and other mammals (21). Anthrax usually occurs following contact with B. anthracis spores, which are formed when growing cells are deprived of certain essential nutrients (5). Sporulation begins when starved cells undergo an asymmetric septation that produces large and small genome-containing compartments called the mother cell and prespore, respectively. The mother cell engulfs the prespore, which is now called the forespore. Three protective layers are formed around the forespore: the innermost region is a thick peptidoglycan layer called the cortex, the middle layer is a proteinaceous coat, and the outermost layer is the exosporium. After a stage of spore maturation, the mother cell lyses and releases the spore (14). Mature spores are dormant and capable of surviving in the soil and other adverse environments for many years (22). When spores encounter an aqueous environment containing appropriate nutrients, they can germinate and grow as vegetative cells. Germination of B. anthracis spores can be activated by small-molecule germinants, which interact with receptors in the spore membrane separating the cortex and the spore core (2, 15).

As the outermost layer of the B. anthracis spore, the exosporium is the primary site of contact with the environment, including host defenses (12). This layer also serves as the source of surface antigens (8, 30) and as a semipermeable barrier that excludes large, potentially harmful molecules such as antibodies and hydrolytic enzymes (8, 9). The exosporium is a loose-fitting, balloon-like structure composed of a paracrystalline basal layer and an external hair-like nap (8, 9). The filaments of the hair-like nap are apparently formed by a single collagen-like glycoprotein called BclA (32), whereas the basal layer is composed of a number of different proteins in tight and loose associations (18, 26, 30). The current list of putative basal layer proteins (Table 1) includes BxpA, BxpB (also called ExsF), BxpC, and ExsK, which are unique to Bacillus species that form spores surrounded by a prominent exosporium (26, 30). The putative basal layer proteins CotY, ExsY, CotB-1, and CotB-2 are homologues of Bacillus subtilis outer coat proteins. CotY and ExsY, which are 84% identical, closely resemble the B. subtilis coat proteins CotY and CotZ (26). CotB-1 and CotB-2, which are 42% identical, resemble the B. subtilis coat protein CotB. The list of putative basal layer proteins also includes three enzymes: alanine racemase, inosine-uridine-preferring nucleoside hydrolase, and superoxide dismutase. The first two enzymes degrade the germinants L-alanine and inosine, respectively, and may function to suppress germination (10, 26, 30, 33). Superoxide dismutase could protect the spore from reactive oxygen species during infection (19), participate in oxidative cross-linking of coat or exosporium proteins (14), or perhaps, as recently suggested, moderate germination efficiency upon exposure to superoxide (1).


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  		TABLE 1. Exosporium proteins of B. anthracis Sterne

None of the putative basal layer proteins listed above has been precisely located within the spore or assigned a structural or functional role. In this study, we examined the location and function of the putative basal layer protein BxpB, which is a 17-kDa protein that was found in purified exosporium preparations by Steichen et al. (30). We show by immunogold electron microscopy that BxpB is indeed located within the basal layer of the B. anthracis exosporium. BxpB forms a high-molecular-mass complex with BclA and probably at least two other putative basal layer proteins. BxpB is required for basal layer attachment of the hair-like nap formed by BclA, and BxpB also suppresses germination.


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MATERIALS AND METHODS
 
B. anthracis strains. The Sterne 34F2 veterinary vaccine strain of B. anthracis, which was used as the wild-type strain in this study, was obtained from the U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Md. The Sterne strain is not a human pathogen, because it lacks a plasmid (i.e., pXO2) necessary to produce the capsule of the vegetative cell (11). Mutant Sterne strains CLT274({Delta}rmlD) and CLT292({Delta}bclA) were previously described (4). Another variant of the Sterne strain that carries a mutation called {Delta}bxpB, which replaces codons 1 to 163 of the 167 bxpB codons with a spectinomycin resistance cassette, was constructed in three steps. First, the {Delta}bxpB mutation was constructed and inserted into the unstable shuttle vector pUTE29 (4). Second, the mutation was recombined into the genome of the Sterne strain by allelic exchange (4). Third, the mutation was moved from the latter strain to a wild-type Sterne background by phage CP51-mediated generalized transduction (11). The final strain was designated CLT307({Delta}bxpB). The mutant locus of this strain was confirmed by PCR amplification of the bxpB region and sequencing the DNA product.

Cloning and expression of recombinant bxpB and purification of the BxpB protein. The bxpB open reading frame of the Sterne strain was amplified by PCR and inserted into the cloning site of the expression vector pET15b (Novagen) as previously described (30). The bxpB-containing plasmid was transformed into Escherichia coli BL21(DE3) to express bxpB according to the pET system manual (Novagen). The expressed recombinant BxpB (rBxpB) protein, with a 6x His tag and a Factor Xa cleavage site immediately preceding the initiating methionine of BxpB, was purified under native conditions by immobilized metal affinity chromatography according to instructions provided by QIAGEN. Briefly, rBxpB in a cell extract was bound to beads of Ni-nitriloacetic acid agarose in native lysis buffer. After binding, the beads were washed five times in native wash buffer, followed by resuspension in Xa cleavage buffer containing 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5 mM CaCl2, and 0.05% sodium azide. Factor Xa protease (Novagen) was added to the slurry of beads, and this sample was incubated at room temperature for 48 h to release native rBxpB into solution. The beads were removed by centrifugation, and Xa was removed by using the Factor Xa Capture kit (Novagen). The protein concentration of the essentially pure rBxpB preparation was determined by using the Bio-Rad protein assay with bovine serum albumin (BSA) as the standard.

Preparation of monoclonal antibodies (MAbs). The mouse anti-BclA MAb EF12 was prepared and characterized as previously described (30). Using purified rBxpB as the immunogen, the mouse anti-BxpB MAb 10-23-4 was prepared essentially as previously described (30). MAbs EF12 and 10-23-4 were both of the immunoglobulin G (IgG) class, with the isotype determined as previously described (16).

Protein gel electrophoresis and immunoblotting. Protein and exosporium samples were boiled for 8 min in 30 µl of sample buffer containing 62.5 mM Tris-HCl (pH 6.8), 2% sodium dodecyl sulfate (SDS), 100 mM dithiothreitol, 0.012% bromophenol blue, and 10% (vol/vol) glycerol. The proteins were then separated by SDS-polyacrylamide gel electrophoresis (PAGE) in 4 to 15% gradient polyacrylamide gels (Bio-Rad) and visualized by staining with Coomassie brilliant blue (23). For immunoblotting, proteins were electrophoretically transferred from an SDS-polyacrylamide gel to a nitrocellulose membrane and treated as described in the manual for the Bio-Rad Immun-Blot Assay kit. Briefly, each blot was blocked with gelatin, probed with the primary MAb at 5 µg/ml for 1 h, and washed. The blot was then probed with a 1:3,000 dilution of goat anti-mouse IgG (heavy plus light chain [H+L]) horse radish peroxidase (HRP)-conjugated secondary antibody for 1 h, washed, and developed with the HRP developer solution.

Preparation of spores and exosporium. Spores were prepared by growing wild-type or mutant B. anthracis strains at 37°C in liquid Difco sporulation medium (23) until sporulation was complete, typically 48 h. Spores were collected by centrifugation, washed extensively with cold (4°C) sterile distilled water, sedimented through a two-step gradient of 20% and 50% Renografin (Bracco Diagnostics), and extensively washed again with cold water (14). Spores were stored and quantitated as previously described (30). Exosporium was purified from spores as previously described (30).

Electron microscopy. All steps in sample preparation were performed at room temperature unless indicated otherwise. For transmission electron microscopy, spores (1010) were fixed for 24 h at 4°C in a solution of 1.25% formaldehyde, 4% paraformaldehyde, and 2% (vol/vol) dimethyl sulfoxide in phosphate-buffered saline (PBS) (28). The spores were thoroughly rinsed in PBS and stained with 1% osmium tetroxide for 3 h at 4°C. After rinsing, the spores were further stained with 1% tannic acid for 20 min and rinsed twice in PBS and once in distilled water. The spores were dehydrated in a graded ethanol series including 50% (vol/vol) ethanol, 70% ethanol containing 1% uranyl acetate (for 1 h), 70% ethanol (twice), 95% ethanol, and 100% ethanol (four times), which was followed by two 30-min rinses in propylene oxide. The spores were embedded in Spurr's low-viscosity resin (Electron Microscopy Sciences). Thin (100-nm) sections of polymerized resin were placed on copper grids and stained with 1% alcoholic uranyl acetate and Reynolds' lead citrate (27) for 9 and 5 min, respectively. Sections were examined under a Hitachi 7000 electron microscope operated at 75 kV.

For immunogold electron microscopy, spores (1010) were fixed for 30 min on ice in a solution of 4% paraformaldehyde and 0.1% (vol/vol) glutaraldehyde in PBS. After two 5-min washes in PBS, the spores were dehydrated in a series of 15-min incubations in 50%, 75%, 95%, and (twice) 100% ethanol. The spores were infiltrated for 1 h in a 1:1 mixture of LR white resin (London Resin Company) and 100% ethanol and then overnight in 100% LR white resin. The spores were infiltrated for 2 h in fresh LR white resin and resuspended again in fresh resin. This suspension was incubated at 42°C for 48 h to embed the spores in polymerized resin. Thin (100-nm) sections of this sample were placed on nickel, carbon-coated Formvar grids. Nonspecific binding sites on the grids were blocked by a 30-min incubation in TSS buffer (20 mM Tris-HCl [pH 7.4], 150 mM NaCl, 20 mM NaN3) containing 1% BSA. The grids were then incubated for 1 h in TSS buffer containing 0.1% BSA and an anti-BclA or an anti-BxpB MAb (10 µg/ml), washed twice with TSS buffer-0.1% BSA, and then incubated for 30 min in a 1:10 dilution (in TSS buffer-0.1% BSA) of goat anti-mouse IgG plus IgM (H+L) labeled with 10-nm gold beads (Amersham Biosciences). The sections were washed in TSS buffer-0.1% BSA and then in PBS, fixed for 2 min in 2% glutaraldehyde in PBS, washed in water, lightly stained with 2% uranyl acetate and Reynolds' lead citrate, and analyzed as described above.

Flow cytometry/fluorescence-activated cell sorting (FACS) analysis. FACS was used to examine the binding of anti-BxpB MAb 10-23-4 and a control (IgG class) MAb to wild-type and mutant spores. Spores (107) were mixed with one of the MAbs at 2 µg/ml in 20 µl of PBS containing 1% BSA, and this mixture was incubated at room temperature for 60 min. Unbound MAb was removed by washing the spores three times in 200-µl volumes of PBS plus 1% BSA. After each wash, spores were collected by centrifugation at 820 x g for 5 min at 4°C. The spore-MAb complexes were then mixed with 20 µl of PBS plus 1% BSA containing 2 µg/ml of Alexa Fluor 488-labeled goat anti-mouse IgG (H+L) secondary antibody (Molecular Probes), and this mixture was incubated at room temperature for 60 min. Unbound secondary antibody was removed by washing spores as described above. The spore-antibody complexes were resuspended in 200 µl PBS plus 1% BSA, and fluorescence was measured by FACS analysis using a FACSCalibur instrument and CellQuest Pro software (Becton Dickinson Biosciences). No spore germination was detected during this assay as judged by microscopic examination.

Preparation of B. anthracis cell extracts. B. anthracis strains were grown in liquid Difco sporulation medium at 37°C with shaking. Beginning at the end of logarithmic growth, 5-ml culture samples were taken hourly for 9 h until sporulation was nearly complete. Cells were harvested by centrifugation at 4,000 x g for 10 min at 4°C, and the pellets were stored at –70°C. Pellets were treated as described above for SDS-PAGE and immunoblotting of protein and exosporium samples.

Monitoring germination and outgrowth. Germination and outgrowth of B. anthracis spores were observed by phase-contrast microscopy using a Leica DM IRBE microscope with a Hamamatsu ORCA-ER digital camera. Time-lapsed photography using Openlab imaging software (Improvisations) was used to obtain direct measurements of morphological changes with time. Spores in water were dried onto a poly-L-lysine-coated 40-mm (diameter) circular coverslip to immobilize them for photography (35). The coverslip was placed in a Bioptechs Focht Chamber System (FCS2) maintained at 37°C by a Bioptechs FCS2 and Objective temperature controller. Prewarmed (37°C) growth medium containing RPMI 1640 1x with L-glutamate (Cellgro, catalog no. 10-040-CV) and 2% brain heart infusion (BHI; Difco) was added to the FCS2 chamber to initiate germination. The germinating spores were observed for a period of 2 h, during which time photographic images were acquired at 10-s intervals.


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RESULTS
 
BxpB is located in the basal layer of the B. anthracis exosporium. To determine the location of BxpB within the B. anthracis spore, we employed immunogold electron microscopy. This technique tags molecules of BxpB within a thin-sectioned spore with a gold bead that is readily detectable by electron microscopy. Tagging requires a primary anti-BxpB MAb and a secondary MAb that is reactive against the primary MAb and also attached to a gold bead. To obtain the primary MAb, we first cloned and expressed the B. anthracis Sterne bxpB gene in E. coli and purified rBxpB to near homogeneity (Fig. 1A, lane rBxpB). This protein was used to immunize mice, from which antibody producing B lymphocytes were obtained and used to make hybridomas. The hybridomas were screened for the production of anti-BxpB MAbs, and one hybridoma producing a MAb designated 10-23-4 was selected for further use. We demonstrated that MAb 10-23-4 could be used as a probe in immunoblots to detect rBxpB (Fig. 1B, lane rBxpB) and BxpB extracted from purified B. anthracis exosporium (Fig. 1B, lane Exo). During gel electrophoresis, most of the detectable BxpB extracted from the exosporium comigrated with rBxpB, while several bands of extracted BxpB migrated more slowly (Fig. 1B, lane Exo). These bands could be BxpB multimers or aggregates of BxpB with different exosporium proteins. In addition, we showed by immunoblotting that MAb 10-23-4 did not react with proteins, presumably including a BxpB paralogue (26), that were extracted from either sporulating cells or mature spores of a Sterne variant unable to produce BxpB due to deletion of the bxpB gene (data not shown).



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  		FIG. 1. Characterization of purified rBxpB and reactivity of the anti-BxpB MAb 10-23-4. (A) Approximately 15 µg of purified Sterne exosporium (Exo) and 1.5 µg of purified rBxpB were separated by SDS-PAGE and stained with Coomassie brilliant blue. (B) The same samples as in panel A were separated by SDS-PAGE, the proteins were electrophoretically transferred to a nitrocellulose membrane, and the membrane was immunoblotted using anti-BxpB MAb 10-23-4 as the primary antibody. Std, molecular mass standards (Bio-Rad).

Using MAb 10-23-4 as the primary antibody and a commercially available goat anti-mouse IgG MAb labeled with 10-nm gold beads as the secondary antibody, we attempted to visualize BxpB on spores of the Sterne strain of B. anthracis by immunogold electron microscopy. On average, 1 to 2 gold beads were found per spore, with a total of 13 spores examined. The beads were always found on the basal layer of the exosporium (Fig. 2A, note that the hair-like nap is not visible due to light staining necessary to preserve epitopes). Although this result indicated that BxpB was a component of the basal layer, the results were not compelling due to the small number of beads bound per spore.



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  		FIG. 2. Localization of BxpB to the exosporium basal layer of Sterne and {Delta}bclA spores. Immunogold electron microscopy, with anti-BxpB MAb 10-23-4 as the primary antibody, was used to tag BxpB in thin sections of Sterne (A) and {Delta}bclA (B) spores. Electron-dense gold beads (10 nm in diameter) indicate the position of reactive BxpB molecules. The filaments of the exosporium hair-like nap, which are present on the Sterne spores shown in panel A, are not visible due to light staining required for immunogold tagging. In each panel, an arrowhead points to the exosporium basal layer. Magnification of the electron micrographs was 30,000x.

One possible explanation for the apparently poor labeling of BxpB was that access to BxpB epitopes was limited by the presence of the native hair-like nap on the exosporium. The nap could block access to BxpB epitopes, or it could cause BxpB to adopt a conformation that reacted poorly with the MAb. Therefore, we examined spores produced by two variants of the Sterne strain that display an altered or no hair-like nap. The first variant was strain CLT274({Delta}rmlD), which is unable to synthesize L-rhamnose, a major component of BclA oligosaccharide side chains. Spores produced by the {Delta}rmlD strain contain a hair-like nap that is glycosylation deficient (4). When {Delta}rmlD spores were examined by immunogold electron microscopy, we found an average of 2 to 3 gold beads per spore, with 12 spores examined (data not shown). Thus, it appeared that reducing the glycosylation of BclA and the hair-like nap only marginally increased access to BxpB epitopes. The second variant was strain CLT292({Delta}bclA), which produces spores devoid of a hair-like nap (4). When {Delta}bclA spores were examined by immunogold electron microscopy, we found an average of 18 gold beads per spore, with 14 spores examined (Fig. 2B). This result indicated that BclA or the hair-like nap formed by BclA was indeed restricting exposure of BxpB epitopes. More importantly, this result provided clear evidence that BxpB was a component of the basal layer. In each of the three immunogold tagging experiments described above, control experiments were performed in which the primary MAb was omitted. Essentially no nonspecific binding of gold beads to spores was observed. Furthermore, binding of gold beads was not detected in an immunogold tagging experiment using mutant spores lacking BxpB (data not shown).

To verify that the native hair-like nap could also limit exposure of BxpB epitopes on intact spores, we used FACS to examine the binding of MAb 10-23-4 to Sterne, {Delta}rmlD, and {Delta}bclA spores (Fig. 3). The results showed weak binding of the anti-BxpB MAb to Sterne spores. Binding of the MAb to {Delta}rmlD and {Delta}bclA spores was increased approximately 2-fold and 15-fold, respectively. This pattern of MAb binding is essentially the same as that observed with thin sections of spores. Thus, two independent methods indicate that BclA and the hair-like nap on spores restrict binding of MAbs to BxpB and that this restriction is due primarily to the protein component of BclA.



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  		FIG. 3. FACS analysis of the binding of anti-BxpB MAb 10-23-4 to Sterne, {Delta}rmlD, and {Delta}bclA spores. In each panel, the boldface line represents the histogram obtained with MAb 10-23-4, and the gray histogram was obtained with a control MAb that does not bind B. anthracis spores. Each panel is labeled with the name of the spore examined.

BxpB and BclA interact in a high-molecular-mass complex. It had previously been shown that alkali extraction of wild-type B. anthracis spores released BxpB, BclA, and ExsY that comigrated during SDS-PAGE as a diffuse band with an apparent molecular mass of >250 kDa (26). This result suggested that BxpB, BclA, ExsY, and perhaps other proteins (such as CotY) formed a large, stable complex in the exosporium (26). We found that >250-kDa complexes containing BclA and BxpB could also be extracted from Sterne spores by boiling in sample buffer. BclA and BxpB were detected in the complexes by SDS-PAGE and immunoblotting with anti-BxpB (Fig. 4A, lane wt) and anti-BclA (Fig. 4B, lane wt) MAbs as probes. It appeared that BxpB was present in only a subset of the high-molecular-mass complexes that contained BclA (compare wt lanes in Fig. 4A and B). This subset (i.e., the broad >250-kDa band in Fig. 4A, lane wt) also contained ExsY and possibly CotY, which was determined by recovering this material from the gel, digesting it with trypsin, and sequencing the tryptic peptides by tandem mass spectrometry to identify proteins encoded by the B. anthracis genome (20) (data not shown). In addition to the high-molecular-mass complexes, a large fraction of the BxpB extracted from wild-type spores migrated as a monomer (Fig. 4A, lane wt) with the same apparent mass as rBxpB (Fig. 1). This result indicated that BxpB was not glycosylated, a possibility suggested by previous studies (30).



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  		FIG. 4. Detection of BxpB- and BclA-containing material extracted from Sterne and {Delta}bclA spores. After boiling in sample buffer, the material extracted from Sterne (wt) and {Delta}bclA spores was separated by SDS-PAGE and electrophoretically transferred to a nitrocellulose membrane in duplicate. One membrane was immunoblotted using anti-BxpB MAb 10-23-4 (A), and the other was immunoblotted using anti-BclA MAb EF12 (B). The gel location of monomeric BxpB in panel A is indicated with an arrowhead. Std, molecular mass standards.

To further study the protein interactions within the >250-kDa complex, we examined BxpB-containing complexes extracted from spores of strain CLT292({Delta}bclA). These spores do not contain BclA, which we confirmed by immunoblotting (Fig. 4B, lane {Delta}bclA). When the {Delta}bclA spores were extracted and examined for BxpB-containing complexes by immunoblotting with an anti-BxpB MAb, no >250-kDa complexes were detected (Fig. 4A, lane {Delta}bclA). This result provided direct evidence that BclA and BxpB were both in the >250-kDa complexes. In the absence of BclA, only faster migrating and presumably smaller BxpB species were detected. Most of the detectable BxpB appeared to be monomers. Another major species with an apparent molecular mass of ~38 kDa could be a dimer of BxpB. Many other minor bands of BxpB-containing material were detected, with most migrating with apparent masses between 50 and 100 kDa. This material may include other exosporium proteins. None of this material was glycosylated, as judged by the absence of staining with the ECL Glycoprotein Detection kit (Amersham) (data not shown). This result provided additional evidence that BxpB was not a glycoprotein.

BxpB is required for the attachment of BclA to the basal layer of the exosporium. Because our results indicated an interaction between BxpB and BclA, we examined the possibility that spores devoid of BxpB would be unable to incorporate BclA and form a hair-like nap. We constructed a mutant Sterne strain, designated CLT307({Delta}bxpB), which contains a deletion removing essentially the entire bxpB gene. Spores of this strain were prepared and examined by transmission electron microscopy. These spores appeared to be devoid of the hair-like nap (Fig. 5A), which was clearly present on Sterne spores that had been prepared identically for inspection (Fig. 5B). Although the spores examined in this experiment had been purified, the purification procedure did not alter the appearance of the spores (data not shown). We then used immunogold electron microscopy with an anti-BclA MAb to examine the {Delta}bxpB and Sterne spores for the presence of BclA. For the {Delta}bxpB spores, no gold beads were found on 36 of the 50 spores examined. On average, we found 0.4 beads per spore, with a range of 0 to 4 beads per spore (Fig. 5C). In contrast, for the Sterne spores we found many gold beads on each of the 50 spores examined. The average number of beads per spore was 29, with a range of 12 to 51 beads per spore (Fig. 5D). Again, note that the hair-like nap is not visible in Fig. 5C and D due to the light staining necessary to preserve epitopes.



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  		FIG. 5. Electron micrographs used to examine the exosporium of {Delta}bxpB and Sterne spores for the presence of hair-like nap and BclA. Transmission electron micrographs (TEM) show the exosporium of {Delta}bxpB (A) and Sterne (B) spores. Immunogold electron micrographs (IEM), which used anti-BclA MAb EF12 as the primary antibody, show 10-nm gold beads attached to reactive BclA molecules on the exosporium of {Delta}bxpB (C) and Sterne (D) spores. In panels C and D, light staining does not permit detection of the exosporium hair-like nap. In all four panels, an arrowhead points to the exosporium basal layer. Magnification of the electron micrographs was 30,000x.

To further investigate the apparent loss of BclA from {Delta}bxpB spores, we used immunoblotting with an anti-BclA MAb to measure BclA levels extracted from the same number of Sterne and {Delta}bxpB spores (from 106 to 109). The results indicated that the amount of BclA extracted from the {Delta}bxpB spores was approximately 2% of that from Sterne spores (Fig. 6). This result is consistent with the interpretation that {Delta}bxpB spores are severely deficient in attaching BclA to the exosporium and forming the hair-like nap. To exclude the possibility that the failure to assemble BclA on the {Delta}bxpB spore was due to an inability to synthesize BclA, we compared BclA synthesis in sporulating Sterne and {Delta}bxpB cells. Beginning at the end of logarithmic growth, culture samples were taken hourly for 9 h until sporulation was nearly complete. Cell extracts were analyzed by SDS-PAGE and immunoblotting with an anti-BclA MAb (Fig. 7). The results showed that the time of appearance of BclA and the levels of accumulated BclA were essentially the same in both strains, as determined by densitometric scanning of the blots (data not shown). The only difference observed between the two strains was that BclA from the Sterne strain migrated in the gel as a broad ≥250-kDa band (Fig. 7A), whereas BclA from the {Delta}bxpB strain migrated as two well-separated bands with apparent molecular masses of ~250 kDa and ~200 kDa (Fig. 7B). The lower molecular mass bands from the {Delta}bxpB strain (also observed with spores in Fig. 6) may reflect an inability to assemble BclA into larger complexes necessary for proper nap formation. In any event, our results clearly indicate that the {Delta}bxpB mutation does not affect the production of BclA but apparently precludes the efficient attachment of BclA to the basal layer of the exosporium.



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  		FIG. 6. Relative amounts of BclA-containing material extracted from {Delta}bxpB and Sterne spores. Samples of each spore, ranging from 106 to 109 spores, were boiled in sample buffer. Extracted material was separated by SDS-PAGE and electrophoretically transferred to a nitrocellulose membrane. The membrane was immunoblotted using anti-BclA MAb EF12 as the primary antibody. The amount of BclA-containing material in each lane was measured densitometrically using a Bio-Rad Gel Doc EQ system with Quantity One software.



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  		FIG. 7. Relative amounts of BclA-containing material in sporulating cells of the Sterne and {Delta}bxpB strains. Equal numbers of Sterne (A) and {Delta}bxpB (B) cells were harvested at hourly intervals, beginning at the end of logarithmic growth (only the 3- to 9-h samples are shown here). These samples were boiled in sample buffer, and the material extracted was separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes (one per strain). The membranes were immunoblotted in parallel using anti-BclA MAb EF12 as the primary antibody. The sizes (in kilodaltons) of molecular mass protein standards are indicated to the right of each blot.

The {Delta}bxpB mutation has no or little effect on cell growth, spore formation, and resistance properties of the spore. To investigate the physiological effects of the {Delta}bxpB mutation, we compared the growth rates, cell yields, and sporulation efficiencies of cultures of the Sterne and CLT307({Delta}bxpB) strains grown in liquid Difco sporulation medium at 37°C with shaking. We observed no significant differences between the two cultures, with both growing with maximum doubling times of ~25 min and sporulating with >95% efficiency (data not shown).

Bacillus spores are resistant to organic solvents, lysozyme, and heat. Using standard protocols (23), we compared the resistance of Sterne and CLT307({Delta}bxpB) spores to these treatments. We found no significant difference in the survival of Sterne and {Delta}bxpB spores treated with chloroform, methanol, phenol, or heat (Table 2). However, the Sterne spores were approximately 1.5-fold more resistant than {Delta}bxpB spores to lysozyme (Table 2), a 14-kDa enzyme that can hydrolyze the peptidoglycan in the spore cortex and underlying germ cell wall (6, 7). This reproducible difference suggests that relatively large molecules move more easily through the exosporium of {Delta}bxpB spores. However, the suggested increase in permeability is small and apparently not associated with a major structural change in the basal layer of the exosporium of {Delta}bxpB spores (Fig. 5).


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  		TABLE 2. Chemical and heat resistance of Sterne and CLT307({Delta}bxpB) spores

{Delta}bxpB spores exhibit a faster rate of germination and outgrowth. We also measured the effect of the {Delta}bxpB mutation on spore germination and outgrowth. Spores of either the Sterne or CLT307({Delta}bxpB) strain were dried onto a coverslip, which was placed in a microscope chamber maintained at 37°C. After allowing the coverslip to warm to 37°C, the chamber was filled with prewarmed (37°C) RPMI-BHI growth medium. This rich growth medium contains multiple germinants, including L-alanine and other amino acids. Spore germination and outgrowth were monitored by phase-contrast microscopy and time-lapsed photography as described in Materials and Methods. The marker for germination was the change in spore appearance from refractile to phase dark (Fig. 8A and B), which occurs upon rehydration of the core and lysis of the cortex (25). The marker for outgrowth was the sudden popping of the vegetative cell from its exosporium "shell" (Fig. 8C to D). Our results showed that, on average, germination of {Delta}bxpB spores occurred approximately 2 min earlier and the outgrowth of {Delta}bxpB spores occurred approximately 15 min earlier than observed with Sterne spores (Fig. 9). Although not shown in Fig. 9B, outgrowth of the Sterne spores continued to parallel the outgrowth of the {Delta}bxpB spores, and all phase-dark Sterne spores eventually grew as vegetative cells (Fig. 8D and E). Essentially the same results were obtained when this experiment was repeated with different preparations of Sterne and {Delta}bxpB spores. Thus, the presence of BxpB in the exosporium appears to suppress germination and outgrowth. The effects of BxpB on germination appeared to be independent of its role in attaching BclA to the exosporium, because the time course of germination and outgrowth of spores of strain CLT292({Delta}bclA) was indistinguishable from that of Sterne spores (data not shown).



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  		FIG. 8. Microscopic visualization of spore germination and outgrowth. To illustrate the morphological changes that occur during spore germination and outgrowth, spores of the Sterne strain were tethered to a coverslip, placed in rich growth medium at 37°C, and monitored by phase-contrast microscopy and time-lapsed photography. Shown are selected photomicrographs focusing on a single spore (marked by an arrowhead) that were taken at the times (in minutes) indicated at the bottom of each picture. These photomicrographs show the phase darkening of the spore that is used to indicate germination (A and B), the popping of the cell from the exosporium (exo) that is used as the marker for outgrowth (C and D), and the elongation of the cell that indicates vegetative growth (D and E). In addition, the swelling of the phase-dark spore, which occurs gradually and precedes outgrowth, is shown (B and C).



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  		FIG. 9. Time course of germination and outgrowth of Sterne and {Delta}bxpB spores. Spores (either Sterne or {Delta}bxpB) were tethered to a coverslip, placed in rich growth medium at 37°C, and monitored by phase-contrast microscopy and time-lapsed photography for morphological changes indicative of germination and outgrowth. The percentages of spores that germinate (A) and the percentages of germinated spores that pop from their exosporium to begin vegetative growth (B) are plotted as a function of time.

A possible explanation for the faster germination of {Delta}bxpB spores was that the loss of BxpB resulted in reduced levels of one or more of the putative exosporium enzymes, alanine racemase, inosine-uridine-preferring nucleoside hydrolase, and superoxide dismutase. To examine this possibility, we extracted surface proteins from Sterne and {Delta}bxpB spores by boiling in sample buffer and analyzed the proteins by SDS-PAGE and Coomassie staining. Bands corresponding to alanine racemase, inosine-uridine-preferring nucleoside hydrolase, and superoxide dismutase were previously identified (26, 30). The intensities of the stained protein bands in the gel indicated that the levels of each enzyme were the same in the two spore extracts (data not shown). In addition, we confirmed by immunoblotting that the levels of alanine racemase and superoxide dismutase in the two spore extracts were essentially identical (data not shown). For these experiments, mouse MAbs were raised against recombinant alanine racemase and superoxide dismutase essentially as described for the production of anti-BxpB MAbs in Materials and Methods. Taken together, our results demonstrated that the faster germination of {Delta}bxpB spores was not due to an altered level of alanine racemase, inosine-uridine-preferring nucleoside hydrolase, or superoxide dismutase.


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DISCUSSION
 
BxpB was discovered as one of several proteins present in a purified sample of exosporium that had been isolated from the Sterne strain of B. anthracis (30). BxpB, with a gel mobility consistent with its predicted monomeric molecular mass, was found in much higher levels in samples of chemically deglycosylated exosporium. This result suggested that BxpB was either glycosylated or associated with glycosylated material (30). The results of this study indicate that BxpB is not a glycoprotein but forms a high-molecular-mass complex with the glycoprotein BclA and other proteins such as ExsY and possibly CotY, which are known or putative exosporium proteins. We used immunogold electron microscopy, with an anti-BxpB MAb as the primary antibody, to confirm that BxpB is an integral basal layer protein. Binding of the anti-BxpB MAb to Sterne spores was weaker than that detected with {Delta}bclA spores that lack BclA. The observed inhibition of MAb binding to Sterne spores was due primarily to the protein component of BclA, perhaps reflecting close BxpB-BclA interactions.

To determine the function of BxpB within the exosporium, we examined {Delta}bxpB spores that lack BxpB. Transmission and immunogold electron microscopy revealed that these spores were enclosed by an exosporium that was devoid of hair-like nap. The inability to assemble a hair-like nap was not due to a lack of BclA, which was present in normal levels in sporulating {Delta}bxpB cells. However, very little of this BclA was associated with {Delta}bxpB spores (i.e., ~2% of the level found on Sterne spores). Therefore, it appears that BxpB is required for the proper attachment of BclA to the basal layer and assembly of the hair-like nap. Recent studies indicate that this attachment occurs through the 38-residue amino-terminal domain (NTD) of BclA, with the rest of the collagen-like BclA protein extending away from the spore (3). The first 19 amino acids of this NTD are absent in mature BclA isolated from spores, apparently due to a proteolytic processing event (30, 32). It is tempting to speculate that this processing event is linked to the attachment of BclA to the basal layer of the exosporium, perhaps through a covalent linkage directly to BxpB. The BxpB-containing high-molecular-mass material extracted from Sterne spores by boiling in sample buffer (Fig. 4A, lane wt) could include this covalently linked material. Accordingly, the monomeric BxpB in this extract could represent BxpB present in the basal layer but unattached to BclA. The alternative view, with equal room for speculation, is that BxpB's role in BclA attachment to the basal layer is indirect.

In light of the fact that deleting the bxpB gene appears to completely prevent the formation of the hair-like nap, it is interesting to note that B. anthracis contains a gene that encodes a paralogue of BxpB (26). This gene is designated BAS2303 in the Sterne genome, and the sequence of the encoded 167-amino-acid protein is 78% identical with BxpB. Apparently, the BAS2303 gene is unable to complement the {Delta}bxpB mutation, indicating separate functions for BxpB and its paralogue. These separate functions may be reflected by different stage-specific promoters that direct transcription of bxpB and BAS2303. The bxpB gene appears to be the second gene of a two-gene operon, namely cotY-bxpB, in which the cotY structural gene is located 39 nucleotides downstream of a sequence (i.e., ATA-16-nucleotide spacer-CATA---T) that strongly resembles the consensus {sigma}E promoter (13). On the other hand, BAS2303 appears to be part of a single-gene operon with the structural gene located 67 nucleotides downstream of the consensus sequence (i.e., AC-17-nucleotide spacer-CATA---T) for a {sigma}K promoter (13). Although both {sigma}E and {sigma}K are active in the mother cell, in the sigma cascade that directs temporal gene expression during sporulation {sigma}E precedes {sigma}K (31). Therefore, it is possible that the absence of BxpB cannot be compensated for by its paralogue, because these two proteins are synthesized at different times during spore development. It should be noted that the protein encoded by BAS2303 has yet to be found in purified exosporium preparations.

Although {Delta}bxpB spores lack a hair-like nap, the basal layer around these spores looks similar to that of Sterne spores in electron micrographs. This observation indicates that the general appearance of the normal basal layer is due to the exosporium proteins retained by {Delta}bxpB spores, which likely include ExsY, CotY, CotB-1, and CotB-2. These proteins are homologues of the B. subtilis outer coat proteins CotY, CotZ, and CotB, which are cross-linked in the mature spore apparently to confer insolubility and chemical and mechanical resistance to the spore coat (14). In the B. subtilis spores, the cysteine-rich CotY and CotZ proteins are cross-linked through disulfide bonds. The B. anthracis homologues of these two proteins, namely ExsY and CotY, are also cysteine rich, and it seems likely that they too are cross-linked through disulfide bonds, but in this case within the exosporium. In B. subtilis spores, the CotB protein, which does not contain cysteines, is apparently irreversibly cross-linked through an unknown mechanism to form CotB dimers and/or complexes with one or more other outer coat proteins (36). Surprisingly, the CotB-1 and CotB-2 proteins of B. anthracis, which are homologues of CotB, contain 8 and 10 cysteine residues, respectively. These large numbers of cysteine residues suggest that CotB-1 and CotB-2 are cross-linked through disulfide bonds in the exosporium. Apparently, CotB-1 and CotB-2 associate with proteins differently than CotB. Although it seems likely that disulfide bonds are involved in the cross-linking of ExsY, CotY, CotB-1, and CotB-2 within the exosporium, reducing agents are not sufficient to disrupt high-molecular-mass complexes containing at least some of these proteins along with BclA and BxpB. Thus, additional and presently undetermined protein associations appear to exist within the exosporium. It should also be noted that the apparently overlapping protein compositions of the B. anthracis exosporium and B. subtilis outer coat suggest similarities in the structure and function of these two layers, an idea that has also been proposed by others (14, 29, 34).

Our results demonstrated a second function for BxpB, which appears to be independent of its role in attaching BclA to the basal layer. We found that germination and outgrowth of {Delta}bxpB spores occurs 2 and 15 min earlier, respectively, than observed with Sterne spores. This result indicates that BxpB retards germination. Perhaps under certain circumstances, this effect could even preclude spore germination. This effect of BxpB is particularly interesting because the B. anthracis exosporium contains the enzymes alanine racemase, inosine-uridine-preferring nucleoside hydrolase, and superoxide dismutase, which also have the potential to preclude germination. It appears likely that the spore possesses these activities to avoid germination, e.g., by degrading low concentrations of germinants, under conditions that are unable to support viable cell growth. We found that the levels of alanine racemase, inosine-uridine-preferring nucleoside hydrolase, and superoxide dismutase are similar in Sterne and {Delta}bxpB spores, indicating that the inhibition of germination by loss of BxpB is not the result of changes in the levels of these exosporium enzymes. Presently, an argument is ongoing about the possibility that B. anthracis spores are uniquely averse to germination and in fact rarely germinate in their soil environment (24). If such an aversion exists, it seems likely that BxpB and the exosporium enzymes contribute independently to this condition.


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ACKNOWLEDGMENTS
 
We thank Leigh Millican of the UAB High Resolution Imaging Facility for invaluable assistance with electron microscopy. Mass spectrometry was performed by Marion Kirk and Landon Wilson in the UAB Comprehensive Cancer Center Mass Spectrometry Shared Facility. We thank Jeremy Boydston and Dakin Williams for their help and advice.

This work was supported by NIH grant AI50566, and C.T.S. was supported by NIH training grant HL07553.


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FOOTNOTES
 
* Corresponding author. Mailing address: UAB Department of Microbiology, BBRB 409, 1530 3rd Ave. S, Birmingham, AL 35294-2170. Phone: (205) 934-6289. Fax: (205) 975-5479. E-mail: ChuckT{at}uab.edu. Back


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Journal of Bacteriology, September 2005, p. 5868-5876, Vol. 187, No. 17
0021-9193/05/$08.00+0     doi:10.1128/JB.187.17.5868-5876.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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